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J i..; - '■ 







■' ■ 1996 

A dedication to my parents and my husband: 
for their love 

* <■■'* 


I would like to thank my mentor, Dr. Noel K. Maclaren, 
for his guidance and insight. I have gained much from his deep 
and broad knowledge of immunology, genetics and molecular 
biology. His encouragement was the main driving force for all 
my success. 

I would like to thank my committee members, Drs. 
Catherine Hammett- Stabler, Joel Schif f enbauer, Jin-Xiong She, 
and especially Dr. Nancy Denslow, for their helpful 
suggestions, discussions and encouragement. 

I am very grateful to Dr. Jeff Anderson for his critique 
on this manuscript . 

I also thank all my friends and collegues at the 
Department of Pathology and Laboratory Medicine. 

My sincere thanks go to my parents and my sisters, for 
all of their love and encouragement in the past years. 

Finally, I would like to thank my husband, Dr.Yao-Hua 
Song, for his love and support. '■-*'''. 



f t !- ; 







Autoimmune Disease 1 

Overview 1 

Classification of Autoimmune disorders 2 

Genetic Factors in Autoimmunity 4 

Etiological Factors in Autoimmunity 5 

Mechanisms of Autoimmunity 6 

Autoreactive T cell 6 

Autoreactive B cell 11 

The Role of Autoantibodies 13 

Methods to Detect Autoantibodies 14 

In vitro Translation 18 

Autoimmune Polyglandular Syndrome (APS) 19 

Type I APS 21 

Type II APS 23 

Enzymes and Receptors as Autoimmune Targets .... 25 

Specific Aims of This Research 27 


Introduction 29 

Materials and Methods 31 

Amplification of Tyrosinase cDNA 31 

DNA Sequencing and Expressing 32 

Results 33 

Discussion 33 



Introduction 3 7 

Materials and Methods 3 8 

Patients 38 

In Vitro Translation and Immunoprecipitation ... 38 

Results 40 

Discussion 40 


Introduction 4 9 

The Anatomy and Physiology of 

Parathyroid Gland 4 9 

The Structure and Function of Calcium 

Sensing Receptor (Ca-SR) 50 

The Detection of Autoantibodies 

in AH in the Past 54 

Materials and Methods 55 

Patients 55 

Antigen Preparation 55 

Immunoblotting 57 

In Vitro Translation and Immunoprecipitation ... 58 
Absorption of Autoantibodies with 

Recombinant Ca-SR 60 

Results 60 

The Identification of Autoantibodies in AH ... . 60 

The characterization of Ca-SR in AH 72 

Discussion 89 


Introduction 97 

Materials and Methods 99 

Antigen Preparation 99 

Immunoblotting 100 

Results 100 

Discussion 103 





1. The Classification of Autoimmune Disease 3 

2 . Characteristics of Autoimmune Disorders in APS I . 22 

3 . The Frequency of Autoimmune Disorders in APS I 

and APS II 24 

4. Autoantibody Reactivity to ZP3 43 

5. Characteristics of AH Patients 56 

6. Autoantibody Reactivity to Recombinant Calcium 

Sensing Receptor 77 

7. Autoantibody Reactivity to In vitro Translated 

Domains of Calcium Sensing Receptor .... 88 

8. Characteristics of Positive AH patients 91 

9. Autoantibody Reactivity to Ca-SR 94 

10. Autoantibody Reactivity to Human Head 

Skin Homogenate 104 



1. PCR Amplification of Human Tyrosinase cDNA 35 

2. • Immunoprecipitation of Human ZP3 42 

3. Immunoprecipitation of Human ZP3 with 

••" Reduced Amount of Detergent 45 

4. Diagram of the Strategy for in vitro Translation 

of Calcium-Sensing Receptor 53 

5. Immunoblot Analysis Using the Cytosolic Fraction of 

Human Parathyroid Gland Extract 62 

6. Immunoblot Analysis of the Cytosolic Fraction of 

Human Parathyroid Gland Extract Using 

Normal Human Sera 65 

7. In vitro Translation of Human Parathyroid 

Secretory Protein (PSP) 67 

8. Immunoprecipitation of Human Parathyroid Secretory 

Protein by Control Sera 69 

9. Immunoprecipitation of Human Parathyroid Protein by 

Sera from Patients with Hypoparathyroidism . . 71 

10 . Immunoblot Analysis with Human Parathyroid 

Gland Extract 74 

11. Immunoblot Analysis Using Membranes of HEK-293 Cells 

Transfected with Human Ca-SR cDNA 76 

12. In vitro Translation of the Extracellular Domain 

of the Ca-SR 80 

13. Immunoprecipitation of the Extracellular Domain 

by Rabbit Anti-Ca-SR 82 

14. Immunoprecipitation of the Extracellular Domain 

by AH Sera 84 

15. Absorption Studies with HEK-293 Cells 87 


16. Immunoblot Analysis Using Human Head Skin 

Homogenate 102 



Abstract of Dissertation Presented to the Graduate School of 
the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 



Yangxin Li 

August, 1996 

Chairman: Noel K. Maclaren 

Major Department: Pathology and Laboratory Medicine 

Type I autoimmune polyglandular syndrome (APS) consists 
of three primary disorders: Addison's disease, chronic 
mucocutaneous candidiasis and hypoparathyroidism as well as 
several commonly associated disorders such as gonadal failure, 
alopecia and vitiligo. The search for specific targeted 
autoantigens involved in APS has been the major focus of my 

Human parathyroid gland was homogenized and cytosolic and 
membrane fractions were obtained by ultracentrifugation. 
Specific autoantigens with molecular weight of 70 and 80 kDa 
were found in the cytosolic fraction and a 120-140 kDa 
autoantigen was localized to the membrane fraction. The 120- 
140 kDa autoantigen has the same molecular weight as the 
calcium sensing receptor (Ca-SR) , therefore, the Ca-SR was 
investigated as a candidate autoantigen. Patient sera were 


tested in parallel with a rabbit anti-Ca-SR antibody in 
immunoblots using a Ca-SR-transfected HEK-293 cell membrane as 
the antigen source. Autoantibodies from patient sera reacted 
to a 12 0-140 kDa protein which was identical in molecular 
weight to that recognized by the rabbit positive control 
antibody. The Ca-SR was then translated into extracellular and 
intracellular domains by an in vitro rabbit reticulocyte 
system. Autoantibodies recognized only the extracellular 
domain. Therefore, the extracellular domain of Ca-SR has been 
identified as the major autoantigen in hypoparathyroidism. 

The sperm receptor ZP3 has been identified as an 
autoantigen in experimental murine autoimmune oophoritis. In 
this study, the human ZP3 cDNA was translated in vitro and 
immunoprecipitated with a monkey anti-ZP3 antibody and sera 
from patients with gonadal failure. The monkey antibody was 
the only one which reacted with ZP3 . Therefore, autoantibodies 
against ZP3 were not found in human patients. Autoantigens 
involved in alopecia were investigated using human head skin 
homogenate as antigen source. Specific autoantigens with 
molecular weights of 46-55, 57 as well as muultiple proteins 
with molecular weight higher than 100 kDa were identified by 

The identification of the autoantigens involved in APS is 
an important step toward the understanding of the pathogenic 
mechanisms of the diseases and it may also lead to the 

development of specific methods for diagnosis as well as 
prevention of the diseases. 

V r 



Autoimmune Diseases 


One of the most important features of the immune system 
is its ability to distinguish foreign antigens from self 
antigens. Normally, each individual's lymphocytes are able to 
recognize and attack foreign invaders, but remain unresponsive 
to self antigens. The condition of unresponsiveness to self 
antigens is called immunological tolerance. The breakdown of 
tolerance to self antigens presumably leads to initiation of 
an autoimmune process which leads to autoimmune diseases. ^"^ 

Autoimmune diseases are estimated to affect as many as 5 
to 7 percent of adults in Europe and North America. In the 
United State alone, 1 to 2 percent of the entire population 
may be affected. This poses a serious women's health issue in 
that two-thirds of all patients with an autoimmune disease are 
women. If autoimmunity can be shown to play a major role in 
atherosclerosis, the leading cause of deaths in the Caucasion 
population, the ratio of the affected population would be much 

■; J V. 


further increased.^'" . ' . 

Historical evidence suggests that autoimmune diseases 
became more common in the 19th century, although this could be 
an artifact of increased recognition.^ Most autoimmune 
diseases are polygenic, whose expression reflects the 
interaction between both genetic and environmental factors 
which contribute to the development of these diseases. ^'^ 

Classification of Autoimmune Disorders 

Autoimmune diseases can generally be placed into two 
categories: organ-specific and non-organ-specific autoimmune 
disease. In organ-specific autoimmune diseases, specific 
organs or tissues are affected. Insulin dependent diabetes 
(IDD) , Addison's disease and Graves' disease are examples of 
diseases in this category.'' Alternatively, non-organ-specific 
autoimmune diseases usually involve multiple organs or 
tissues. Systemic lupus erythematous (SLE) and rheumatoid 
arthritis are in this category.^ The autoantigens in organ- 
specific autoimmune diseases are often unique to the targeted 
tissues, and include tissue specific membrane receptors, 
enzymes and secreted hormones (Table 1) .^ The autoantigens in 
non-organ-specific disorders are usually found ubiquitous in 
all parts of the body include collagen, DNA and histone.^"'^^ 

Table 1. The classification of autoimmune diseases. 

Organ- specific 


Target Antigen 

Addison's Disease 

Adrenal cortex 

21 -hydroxylase 

hemolytic anemia 

Red blood cell 




Parathyroid Gland 

Caelum- sensing 

Chronic active 


P450IID6 1 

Graves' disease 


TSH receptor 

Hashimoto disease 


T3,T4,TSH and 

Hypogonadi sm 

Leydig cells 

P450SCC and 
17a -hydroxylase 

Insulin dependent 

Pancreatic islet 

GAD65 and GAD67, 



Multiple sclerosis 

Brain and spinal 

Myelin basic 

Myasthenia gravis 


ACTH receptor 

Pernicious anemia 

Gastric parietal 

H,K-ATPase and 






Non- organ 


Connective tissue 

Type II collogen 

Systemic lupus 

Kidney, skin, 

DNA,histone 1 


Genetic Factors in Autoimmunity 

Based on the rates of familial transmission, genetic 
predisposition is an important factor in the development of 
autoimmune diseases/ It is now believed that multiple genes 
may contribute to the induction of autoimmunity.^ The major 
histocompatibility complex (MHC) genes are associated with 
most autoimmune diseases such as IDD.^^"^* The probable reason 
for this correlation is that MHC molecules play key roles in 
antigen presentation, T cell selection and activation.^ 
Individuals who carry particular HLA haplotypes such as 
DRB1*03/DQB1*0201 and/or DRB1*04/DQB1*0302 are more likely to 
develop type I diabetes. However, not all individuals develop 
autoimmune diseases even though they carry disease-associated 
HLA alleles. ^^ Therefore, a particular HLA allele itself is 
not sufficient to cause autoimmune diseases. A triggering 
factor such as viral infection or other environmental factors 
appears also to be required to initiate or induce the 
development of the autoimmune process.^ 

In addition to the research on the role of MHC, some 
research groups have focused on T cell receptor (TCR) genes, 
since T cells play a major role in autoimmune process. Even 
though a few studies have shown that TCR genes contributed to 
autoimmune diseases, ^^'^^ no strong evidence supporting the 
association between specific TCR haplotypes and autoimmune 


disorders has yet been clearly proven.^' 

Etiological Factors in Autoimmunity v • ■ 4 ^ ) _ '.^f 

1. Under normal conditions, proteins of certain tissue 
such as the lens of the eye are concealed from the immune 
system. However, once the tissues become damaged, the released 
proteins may provoke an autoimmune response because tolerance 
to them had not been established. 

2. Hormonal influences are also important in certain 
autoimmune diseases. Females are much more prone to hormone 
influence than males, and tend to have much higher rates of 
autoimmune diseases.* 

Howard S.Fox of Scripps has shown that the female hormone 
estrogen can stimulate the transcription of gamma- interferon 
(IFN-y) . IFN-7 is a cytokine that can increase the expression 
of MHC class II by antigen presenting cells, induce the 
production of other cytokines produced by THl cells and 
activate macrophages.^" Thus, estrogen could influence the 
autoimmune process by increasing the production of IFN-7.^^ 

3. Viral and bacterial infection may be associated with 
autoimmunity. ^^"^^ A good example of this is rheumatic fever in 
which the inflammation of the heart and joints follow a 
streptococcal pharyngitis. The autoimmune lesions in the heart 
and its valves are not due to the bacteria itself but rather 

due to the host's immune responses to it. *"" 

It should be noted that microorganisms may induce 
autoimmune diseases not only through molecular mimicry, ^*"^^ 
but also through other means such as the release of 
superantigens, ^^"^^ shift in the spectrum of cytokine 
production^" and the release of sequestered antigens .^^"^* 

Mechanisms of Autoimmunity 

Since class II MHC-restricted helper T cells play a 
central role in all acquired cellular and humoral immune 
responses, while central T cell clonal deletion or peripheral 
anergy are normally effective ways of maintaining tolerance to 
self antigens, it is believed that autoreactive T cells play 
major roles in the autoimmune process .^^"*° Even in the known 
autoantibody-mediated disorders, the defect depends on the 
appearance of autoreactive TH2 lymphocytes, which are required 
for the production of high-affinity autoantibodies.*^ 

Autoreactive T Cells 

Since the variable genes encoding T cell receptors that 
may recognize self antigens are present in the germ line, all 
individuals have the potential to develop an autoimmune 
reaction. Therefore, mechanisms to prevent the maturation of 

r -» '4, J » ^ » •■ •» 


autoreactive T cells are necessary. Fortunately, there are 
such mechanisms present in normal individuals/^'*^ Autoreactive 
T cells to protein antigens that are present in the thymus are 
deleted by negative selection.'**"*^ T cell tolerance to protein 
antigens that are not present in the thymus is induced in the 
periphery /'''^^ 

Autoimmunity may develop if autoreactive T cells escape 
the negative selection process and are allowed to mature and 
leave the thymus. ^^ Autoimmunity may also develop if 
peripheral tolerance fails and inadequately anergized T cells 
are activated by polyclonal activators, sequstered antigens, 
or by cross-reaction between self antigens and infectious 
pathogens. ^^'^* 

1. Failure of self -tolerance in thymus. 

The process which eliminates or inactivates potentially 
autoreactive T cell clones in the thymus is called negative 
selection. ^^'^'' 

The elimination of autoreactive T lymphocytes in the 
thymus is induced by the expression of self antigen by the 
thymic epithelium. If immature T cells in the thymus bind 
antigen via either class I or class II MHC molecules with high 
affinity, the immature T cells are killed by apoptosis or 
rendered functionally inactive .^^'^' Autoreactive CD8* T cells 
are eliminated via class I dependent antigen presentation and 
CD4* T cells via class II dependent antigen presentation. 

" r% 

-Hpi '^ " «- 

Functional inactivation in the thymus also can be induced by 
the lack of a second signal on the antigen presenting cells, 
or by insufficient levels of antigens present in the 
thymus .■*^'^° In order to have negative selection take place, 
the self-antigen must be present in sufficient quantity in the 
thymus. It is possible that some circulating macrophages 
collect cell debris, process and present these materials in 
the thymus, even though they are not actually created there. 
Self -tolerance mechanisms in the thymus may be disrupted 
in many different ways. First, the expression of particular 
MHC alleles may influence clonal deletion of autoreactive T 
lymphocytes by the strength of the affinity of peptide 
associated with the MHC binding groove . ^^ Second, some 
sequestered self -antigens may not reach the immune 
system/thymus during the development of tolerance which occurs 
during late fetal or early neonatal life in mammals. Third, 
defects in apoptosis genes (such as Fas or Fas ligand genes) 
may lead to inadequate clonal deletion. It has been shown that 
the autoreactive thymocytes escape from apoptosis in the 
thymus, then go to the periphery in Ipr mice.^^ Other studies 
argue against the role for Fas in clonal deletion in the 
thymus, but support a role of Fas -mediated apoptosis in 
peripheral tolerance." Fourth, the concentration of self 
peptides in the thymus may play a role in clonal deletion. It 
has been suggested that any peptide that is present 

continuously in the thymus at a concentration greater than 
10/iM reliably deletes reactive T cells unless for some reasons 
the mechanism is defective/^ 

2. Failure of self -tolerance in periphery. 

It is unlikely that all self antigens can be presented in 
the thymus, therefore, alternative mechanisms are required to 
render autoreactive T lymphocytes which have escaped thymic 
negative selection unresponsive. Clonal anergy is the major 
process for inducing self -tolerance in periphery, especially 
for antigens present only in peripheral tissues and not in the 
thymus. If T cells recognize an MHC-associated antigen on 
antigen presenting cells (APCs) that do not present the 
necessary costimulators (eg, B7) for T cell activation, the T 
cells are placed into a stage of anergy where they can become 
unresponsive to subsequent stimulation by the same antigen in 
the presence of costimulators.^*-" The interaction between 
B7.1 and CD28 molecules is required for T cell activation. It 
has been shown that APCs lacking B7 on their surfaces can 
induce T cell anergy. • ' ' 

Self tolerance in periphery can also be disrupted by 
several immunological mechanisms. 

(1) . Sequestered antigen. 

Some sequestered antigens may be expressed as apparently 
"new" antigens as the result of trauma. Alternatively, some 
self antigens may be partially degraded and lead to "new" 

T >•• ^ !Bl t 

10 .• ; ; 

(i.e, cryptic) antigenic targets for the immune system. 
Autoimmune disease may develop if the new antigens are 
recognized as foreign.^* 

(2) . Molecular mimicry. 

Peripheral self -tolerance can also be disrupted through 
the concept of molecular mimicry. ^''■^^ Molecular mimicry is 
defined by a structural homology in a linear amino acid 
sequence between self and invading pathogen (e.g., viral or 
bacteria proteins) . This situation is primarily applicable to 
peptide-specific Thl cells and cytotoxic T cells (CTLs) rather 
than to antibodies, since the latter tend to recognize 
conformational determinants. However, antibody reactivities to 
linear peptides shared by self and foreign molecules have also 
been reported. 

(3) . Polyclonal activation. 

Polyclonal activation refers to T cell activation by 
superantigens. Antigens that bind to all TCRs utilizing a 
specific VS gene segment, regardless of the Va gene segment, 
have been called superantigens. Superantigens stimulate a 
large percentage of all T cell clones at one time. In this 
process, autoreactive T cells which escaped the negative 
selection but are held in an anergized state in periphery, may 
be activated by such means. "-^^ They may proliferate upon 
subsequent encounter to a self antigen, and lead to an 
autoimmune process. A recent study from Schif fenbauer et al.^' 


has shown that the superantigens staphylococcal enterotoxins 
(SEs) B and A are able to induce relapse of EAE in PL/J mice 
by reactivating the autoreactive T cells. The authors also 
proposed an important hypothesis that autoimmune disease is 
induced by a "two-hit" process, i.e, the autoreactive T cells 
are first stimulated through a molecular mimicry mechanism 
after an infection. Autoimmune disease may not develop at this 
stage due to either insufficient numbers of autoreactive T 
cells or a supression mechanism. However, the clinical 
manifestation of the autoimmune disease will develop after a 
second infection with a superantigen-producing organism. 

Autoreactive B Cells 

Certain autoantigens are formed by polysaccharides and 
lipids, but these kinds of antigens are not recognized by MHC- 
restricted T cells. B cell tolerance may be the principal 
mechanism for unresponsiveness to such self antigens." 
Compared to T cell tolerance, B cell tolerance is more 
difficult to induce and requires more antigen. ^^-^^ 

There are two principle mechanisms of B cell tolerance 
induction: clonal deletion and clonal anergy.'"'"''^ 

Clonal deletion may be induced when B cells mature in the 
bone marrow and encounter self antigens,''^ perhaps at the 
stage when they express only the IgM form of membrane 

receptors for antigens. Using double -transgenic mice, 
Christopher C. Goodnow et al." showed that B cell tolerance 
to lysozyme was due to functional inactivation or clonal 
anergy, rather than clonal deletion of self -reactive B cells. 
This finding suggests that clonal deletion plays a minor role 
in B cell tolerance, however this termination could be antigen 
dependent . 

Clonal anergy may be induced by an antigen-receptor 
interaction or by lack of help from T cells, which can block 
the expression of membrane Ig before B cells mature to a stage 
of functional competence.''^ The net result of clonal anergy 
is that autoreactive B cell are often present in normal 
individuals but are functionally unresponsive to antigenic 

The failure of B cell tolerance may be explained by 
several hypotheses. First, anergized B cells can be induced to 
secrete autoantibodies by polyclonal activators, such as 
lipopolysaccharide (LPS) or Epstein-Barr virus (EBV) , which 
function independently of membrane Ig."-'^ Polyclonal B cell 
activation has been considered to be a contributing or 
initiating mechanism of autoimmunity, particularly in systemic 
autoimmune disease, such as SLE.'^''"^^ Second, the activation of 
self-antigen specific T cells which have escaped clonal 
deletion in the thymus can stimulate self-antigen specific B 
cells which are normally present but unresponsive to these 

antigens since they lack T cell help. In this case, the 
autoantibodies may be specific to one or a few related 
antigens, and may lead to organ- specif ic autoimmune diseases. 

The Role of Autoantibodies 

In autoimmune disease, autoantibodies may be the agents 
causing the disease, the consequence of tissue damage, or 
simply indicators of an autoimmune process. The role of 
autoantibodies in the pathogenesis varies with the location of 
the targeted antigen.^" 

(1) Autoantibodies directed against cell surface targets, 
such as hormone receptors, play a major role in autoimmune 
disorders. One example is that anti-acetylcholine (Ach) 
receptor in myasthenia gravis (MS) .^^"®* Another example is 
that ant i- thyroid stimulating hormone (TSH) receptor in 
Graves' disease .^^'®* 

(2) Those autoantibodies directed against extracellular 
targets, such as circulating hormones or extracellular matrix, 
may or may not cause any damage . ^° 

(3) Those apparently directed against intracellular 
targets are usually not pathogenic.^" However, the 
autoantibodies against DNA and histone are pathogenic in SLE. 

While a few types of autoantibodies appear to be involved 
in the actual pathogenesis of autoimmune disease. 

■ ■»' ' V 

autoantibodies arising to self antigens provide important 
hallmarks of autoimmune diseases affecting specific organs or 
tissues. Autoantibodies can usually be detected long before 
the clinical onset of their associated diseases. The detection 
of the autoantibodies can therefore be useful for specific 
clinical diagnostic purposes, as well as for prediction and 
thereby for possible prevention of disease. In general, the 
detection of autoantibodies can be used to support an 
autoimmune etiology and to screen individuals at risk of 
developing autoimmune disease before their onsets of clinical 
syndrome. In pregnant women, the detection of certain 
autoantibodies may predict disease in their fetuses and 
newborns . 

Methods to Detect Autoantibodies ■ 

■ The most commonly used methods to detect autoantibodies 
include indirect immunofluorescence," enzyme-linked 
immunosorbent assay (ELISA),^^ radioimmunoassay (RIA),*^ 
western blot, and immunoprecipitation.^"-'^ 

1. Indirect immunofluorescence. 

Patients sera that contain putative autoantibodies are 
allowed to incubate with a frozen section of the target 
tissue, e.g., pancreas, and the residual sera are then washed 
away with PBS (phosphate-buf f ered saline) . The binding of 

autoantibodies to the section can be detected by the 
subsequent incubation of a secondary antibody labelled with a 
fluorescent dye such as FITC. After washing, the stained 
antigens are visualized by a fluorescence microscope equipped 
with a ultra violet light source. This technique is very 
sensitive and can provide information on the localization of 
the antigens within a tissue. 

Indirect immunofluorescence is currently being used as a 
routine tool to detect autoantibodies against antigens in 
various tissues such as adrenal cortex, gonad, pancreatic 
islet, gastric mucosa, and thyroid. The detection of these 
autoantibodies has been very useful in the diagnosis of organ- 
specific autoimmune diseases such as Addison's disease, 
hypogonadism, IDD, pernicious anemia and autoimmune thyroid 

2. ELISA. 

The antigen used in this method must be soluble and able 
to be coated onto the wells of a 96 -well microtiter plate. 
Bovine serum albumin is then added to the wells to block non- 
specific binding sites. Diluted patient sera are added and 
incubated with the antigen. After washing, a secondary 
antibody conjugated with an enzyme is added. After washing 
again, the binding of the autoantibodies to antigens is 
visualized by adding a soluble substrate to develop a color 
reaction. The titer of autoantibodies is determined by 

spectrophotometer and the intensity of the color is 
proportional to the concentration of autoantibodies. ELISA is 
an extremely rapid technique which is relatively inexpensive, 
therefore, it has been used for initial screening of large 
number of samples. However, some conformation -dependent 
antigenic epitopes may be lost upon the binding of the antigen 
to the plastic wells and autoantibodies to such antigens will 
not be detected. 

3. Western blot. 

The technique of transferring proteins from gel to 
membrane and the subsequent incubation with an antibody is 
called Western or immunoblotting. This technique has been used 
extensively in the detection of tissue autoantigens that react 
to autoantibodies by their linear epitopes. The tissue 
autoantigen has to be solubilized first and then subjected to 
size separation by sodium dodecyl sulf ate-polyacrylamide gel 
electrophoresis (SDS-PAGE) . The separated proteins are then 
transferred onto a membrane such as Immobilon-P or nitro- 
cellulose. Diluted patient sera are then incubated with this 
membrane. Autoantibodies affixed to the antigen-containing 
membrane are detected by the addition of a secondary antibody 
such as a goat -anti -human IgG coupled with an enzyme such as 
alkaline phosphatase. Water-insoluble substrate is then added 
to the membrane to visualize the specific antigens recognized 
by the autoantibodies. 


Immunoblotting is often performed after an autoantigen 
has been demonstrated by immunofluorescence . This technique 
allows the detection of specific molecular weight antigenic 
proteins. Knowledge of the molecular weight of the antigen 
will assist in further characterization of the antigen. Since 
immunoblot usually involves the separation of protein under 
denaturing conditions, it would avoid the problems of 
solubilization, aggregation, and co-precipitation of other 
proteins. However, conformational epitopes may not be detected 
by this method. Polyclonal antisera such sera from patients 
usually give higher background than monoclonal antibodies. 
This problem can be solved by including normal control sera in 
parallel to the patient sera in the blot. 

4. RIA. 

The technique was developed by Yalow and Berson in the 
late 1950s. After years of development, the methodology is now 
fairly simple and rapid. This technique requires the use of 
highly purified antigen. The antigen is usually labelled by 
^^^I and an aliquot of the labelled antigen is incubated with 
patients sera. The antibody-antigen complex is precipitated by 
adding protein A-Sepharose. After washing the unbound antigen, 
the concentration of the autoantibodies can be quantitated by 
counting the radioactivity of the antibody- antigen complex in 
a scintillation counter. 


5. Immunoprecipitation. 

This technique is used to isolate the autoantigen in 
mixture by using an autoantibody directed against this 
autoantigen. Sera are added to ^^S-labeled autoantigen. After 
incubation, protein A-sepharose is added and centrifugation is 
performed. This permits separation of the autoantibody-bound 
autoantigen which is present in the pellet and the unbound 
autoantigen which stays in the supernatant. The labelled 
antigen is then separated from the bound antibody by SDS-PAGE 
and visualized by autoradiography. This detection documents 
that the respective antibody must have been present in the 
patient sera. 

In Vitro Translation 

In vitro translation is a technique to express cloned 
genes in a cell -free system. Currently, rabbit reticulocyte 
lysate or the wheat germ system is being used."-'^ In each of 
these systems, mRNA is translated into protein in the presence 
f a radioactively labeled amino acid such as ^^S-labelled 
thionine. The translated protein is usually in its native 
form and suitable for immunoprecipitation. The wheat germ 
system has been shown to be better suited for the translation 
of prokaryotic and plant mRNAs, whereas the reticulocyte 
system has been used for all other eukaryotic mRNAs. 



t»j • , 

Endogenous mRNAs have been eliminated in both systems by 
incubation with calcium activated nuclease. Therefore, 
background proteins are minimal in these systems. 

In vitro translation can be used to identify 
translational products of mRNAs, to determine the 
translational efficiency of a particular mRNA and to study the 
mechanisms of protein synthesis. 

Canine pancreas microsomal membranes are usually used to 
study cotranslational processing of proteins such as signal 
peptide cleavage, membrane insertion, translocation and 
glycosylation. ' • 

Autoimmune Polyglandular Syndromes (APS) 

The APSs are characterized by the simultaneous occurrence 
of multiple autoimmune endocrinopathies and skin disease. APS 
can be classified into at least two groups based on the 
patients' ages of onset, and their associations with specific 
endocrine disorders and HLA phenotypes . ^*'" 

The autoimmune nature of these diseases has been 
determined based on the presence of lymphocytic infiltration 
in the affected gland, organ specific autoantibodies in the 
serum, cellular immune defects and associations with HLA 
genes. ^^ In contrast to systemic autoimmune diseases, the 
autoantigens of the polyendocrinopathies are organ specific. 

Therefore, APS and systemic autoimmune diseases have distinct 
clinical features that rarely overlap. 

The pathogenic role of autoantibodies against certain 
cellular receptors is well established, whereas those against 
intracellular enzymes are not . Many of the autoimmune 
endocrinopathies appear to be directly mediated by 
autoreactive CDS cytotoxic T cells (CTL) with help from Thl 
CD4 cells, while autoantibodies to cellular receptors which 
arise as part of a Th2 response may directly induce others. 
Mixed cellular and antibody autoimmune responses in any single 
disease are probably pathogenic to some degree.' 

It is not known why the individual components of the APS 
cluster together. One possible explanation is that multiple 
and interactive genetic defects affect immunological 
tolerance. Another possible explanation is that endocrine 
glands may share common antigenic determinants such that 
autoimmunity against a single antigen must result in a process 
associated with involvement of other glands. 

As an autoimmune endocrinopathy progresses, some 
immunological markers may disappear while others may appear. 
Autoantibody markers may also disappear due to elimination of 
the autoantigens which drive the response. 

21 , . , » . 

Type I APS 

> . • * *- ' 

APS I is defined by the occurrence of at least two of the 
three diseases: chronic mucocutaneous candidiasis, acquired 
hypoparathyroidism, and autoimmune (autoantibody-positive) 
Addison's disease. These diseases tend to appear in the order 
of mucocutaneous candidiasis, acquired hypoparathyroidism, and 
Addison's disease.^''"" If one of these three disorders is 
bypassed, it may not ever develop. Acquired hypoparathyroidism 
is the most common endocrinologic manifestation of type I APS, 
occurring in more than 80% of patients. 

Type I APS is a relatively rare syndrome and usually 
presents early in infancy. Males and females are equally 
affected. APS I occurs either as an autosomal recessive 
disease, or as a sporadic disorder. APS I is not linked to 
genes within the HLA-DR genetic region of chromosome 6,"° the 
responsible gene has been recently mapped to chromosome 

Type I APS is also associated with other autoimmune 
diseases, such as early onset pernicious anemia, chronic 
active hepatitis, alopecia, vitiligo, malabsorption syndromes 
and gonadal failure (Table 2) . Pernicious anemia and 
hypogonadism tend to occur last . Pernicious anemia occurs 
especially among older patients. Hypogonadism affects 50% of 
females but is diagnosed less commonly among males with type 


Table 2 . Characteristics of autoimmune disorders in 

APS I . . , . . 


Mean Age of 

Extreme Ages 

Acquired Hypo- 

7.5 yr 

3 mo - 22 yr 
(between 2 yr 

10 yr in 80%) 


5.5 yr 

2 mo - 21 yr 
(between 2 yr 

7 yr in 80%) 


13 yr 

1.5 yr - 34yr 
(between 5 yr 

16 yr in 80%) 



4 yr - 21 yr 


8 yr 

1.5 yr - 3 0yr 


8 yr 

1 yr - 21 yr 


16 yr 

7 yr - 21 yr 


17 yr 

5 yr - 21 yr 


17 yr 

15 yr - 18 yr 


3 yr - 28 yr 


6 yr - 24 yr 

' 23 ' 

I APS. . „, . 

Alopecia and vitiligo may be seen in both APSs, and often 
the degree of these skin lesions is striking. Alopecia 
universalis where there is eventually an absence of all body 
hair is most often seen with type I APS. Difficulties with 
malabsorption are common in APS I, resulting from a variety of 
causes. Chronic active hepatitis is common among type I APS 
patients, and all patients should be screened routinely for 
this problem whenever type I APS has been diagnosed. 

Type II APS 

Type II APS is defined by Addison's disease plus an 
autoimmune thyroid disease and/or IDD. Type II APS usually 
presents later in adult life, and displays a female 
predominance which is often multigenerational .^* 

Type II APS is associated with other autoimmune diseases, 
such as late onset pernicious anemia, vitiligo, celiac 
disease, and myasthenia gravis (Table 3) . Although a few 
individuals with type II APS have been reported to have 
gonadal failure, which is associated with high levels of 
circulating pituitary gonadotrophins, its occurrence is at 
least considerably less frequent than in type I APS. The 
occurence of chronic active hepatitis in type II APS is rare 
in contrast to its high frequency in type I APS. 


Table 3 . The frequency of autoimmune disorders in APS I 
and APS II. 




Addison's Disease 



Hypoparat hyroidi sm 




Thyroid Disease 



Insulin Dependent 



Chronic Active 







Pernicious Anemia 



Gonadal Failure 







25 ' " _ 

Most of the component diseases of APS II are strongly and 
primarily associated with DRB1*03/DQB1*0201 and secondarily 
with HLA-B8 through linkage disequilibrium.'*'^^ These diseases 
are Addison's disease, IDD and Graves' disease. Only 
pancreatic beta cell autoimmunity and IDD are associated with 
DRB1*04/DQB1*0302 . 

Receptors and Enzymes as Autoimmune Targets 

Receptors and enzymes have been identified as the 
targeted autoantigens in a number of autoimmune 
endocrinopathies . 

There are a number of receptors that are autoantigens 
targeted by an immune response in organ- specif ic autoimmune 
diseases. ^°^ These include the thyroid stimulating hormone 
(TSH) receptors in autoimmune thyroid disease/^ acetylcholine 
(ACh) receptors of skeletal muscle in myasthenia gravis/^"" 
gastrin receptors in pernicious anemia/"^ corticotropin 
receptors in Addison's disease/"* and insulin receptors in 

The mechanisms for the involvement of receptors in 
autoimmune responses are probably complex. The first 
possibility is that autoantibodies against cell surface 
receptors may lead to functional abnormalities of the cells 
expressing them, resulting in receptor-mediated stimulation or 

26 . 
inhibition of the targeted cells. One example of stimulation 
by an agonist autoantibody mimicking a physiologic molecule is 
Graves' disease which is caused by the binding of autoantibody 
to TSH receptors such that they are stimulated but in a more 
prolonged manner than for TSH itself (long acting thyroid 
stimulator or LATS) .^^'^^ One example of inhibition by 
antagonist autoantibody is myasthenia gravis which is caused 
by the binding of antibody to ACh receptors. ^^"^^ The second 
possibility is that the autoantibodies cross-link the 
receptors and increase the rate of their degradation which 
ultimately leads to their depletion. The third possibility is 
that the autoantibody can bind to the receptor, fix 
complement, and thereby induce damage to the cells expressing 
the receptor. The remaining feature is that a cellular immune 
response often occurs as accompaning the autoantibody 
response, and lead to cell mediated lysis and target cell 

A number of autoantigens have been found to be 
intracellular enzymes, e.g., IVof-hydroxylase"^ and 21- 
hydroxylase'"'"« in Addison's disease, thyroid peroxidase in 
autoimmune thyroiditis, H,K-ATPase in autoimmune gastritis,^" 
tyrosinase in autoimmune vitiligo, ^^° and glutamate 
decarboxylase in IDD.^^^ 

The mechanisms for involvement of intracellular enzymes 
in autoimmune responses are unknown. It is also interesting to 

note that some of these autoantibodies recognize the catalytic 
site of the target enzyme. IVot- hydroxylase and 21 -hydroxylase 
are such examples. It is unclear how antibodies have access to 
intracellular enzymes in vivo or how adrenal cell surface 
autoantigens identifed by immunofluorescence are related to 
those in the cytoplasm. Peripheral blood T-cell response to 
thyroid peroxidase and glutamic acid decarboxylase peptides 
have been described in Hashimoto disease and IDD, 
respectively. These findings raise the alternative possibility 
that production of autoantibodies against enzymes is a 
secondary phenomenon, the initiating event being T-cell 
mediated cytotoxicity directed against endogenous, enzyme- 
derived peptides co-expressed with MHC molecules on the target 

Specific Aims of This Research 

The goal of this research is to characterize and identify 
the autoantigens in APS by employing immunoblotting, 
immunoprecipitation and eukaryotic expression systems. My 
study was designed with the following specific aims: 

1. To amplify a potential candidate autoantigen (the 
tyrosinase) cDNA in autoimmune vitiligo by PCR for the purpose 
of expression in E.coli, and test the expressed product for 
autoantibody reactivity. 

■ ..-spsf^rir; 


2 . To confirm the autoimmune nature of acquired 
hypoparathyrodism and to identify the autoantigens involved. 

3. To investigate the possibility of ZP3 as a potential 
autoantigen in human gonadal failure. ■ . ' 

4. To characterize the nature of the autoantigens 
involved in alopecia. 



Human vitiligo is a common skin disorder characterized by 
areas of depigmentation due to loss of melanin- forming cells 
or melanocytes. ^^^ Whereas the etiology of vitiligo is 
generally not known, it is often associated with one or more 
autoimmune endocrinopathies such as IDD, Addison's disease. 
Graves' disease, and Hashimoto thyroiditis.'* In addition, 
ant i- melanocyte antibodies which can lyse cultured human 
melancotyes by both complement activation and antibody- 
dependent cellular cytoxicity, in patients with vitiligo, as 
well as in chickens affected by the disease have been 
reported. ^^^"^^^ 

Using cultured melanoma cell lines and cultured human 
melanocytes, Naughton et al^"'^^'' demonstrated the occurrence 
of anti-melanocyte antibodies in patients with vitiligo by 
immunofluorescent staining and immunoprecipitation methods. 

Vitiligo has also been studied extensively in the Smyth 
Chicken model. Melanocyte autoantibodies were detected in the 
sera of affected chicks several weeks prior to the expression 
of depigmentation, and the autoantibodies identified were 


shown to bind to multiple melanocyte proteins of between 65 to 
80 JcDa.^^^ 

The key enzyme involved in melanin synthesis is 
tyrosinase with a calculated molecular weight of 62 kDa. 
However, the native form of tyrosinase has a molecular weight 
of approximately 70 kDa.^^^ Tyrosinase has been identified to 
be an important T cell target in melanoma. Visseren et al^^° 
reported that tyrosinase specific autoreactive CTL precursors 
are present in the blood of healthy donors which can be 
activated in vitro by exposure to a synthetic nonapeptide (AA 
369-377) of tyrosinase. It was suggested that autoreactive 
CTLs specific to tyrosinase can best be activated, when the 
antigens are presented in high amounts such as in the case of 
melanoma . 

Since patients with vitiligo have the loss of 
integumentary melanocytes, the aim of the research is to 
explore the possibility that a key enzyme or enzymes involved 
in melanin synthesis could be important autoantigens in the 
disease pathogenesis. My role in this study was to amplify the 
tyrosinase cDNA by PCR for the purpose of expression in 
E.coli . 


Materials and Methods 

Amplification of Tyrosinase cDNA 

The original tyrosinase freeze -dried clone was purchased 
from American Type Culture Collection (ATCC, Rockville, MD) . 
The E.coli clone was grown up in Luria-Bertani (LB) culture 
medium containing ampicillin and the tyrosinase-containing 
plasmid was purified by a mini -prep kit from Promega (Alkaline 
lysis methods) . 

The purified plasmid was then used as template for PCR. 
Two primers were used to amplify the full-length human 
tyrosinase cDNA for expression as fusion proteins with 
glutathione S-transferase (GST) . The primers were designed 
with Bam HI and Eco Rl restriction sites on their 5' end and 
3' ends respectively for directional subcloning. PCR was 
performed using a Perkin Elmer Cetus DNA thermal cycles in a 
total volume of lOO/il. Each cycle consisted of 40 seconds 
denaturation at 94°C, 1.5 minutes annealing at 55°C and 3 
minutes of chain extension at 72°C. Following PCR, the 
overlaying oil was removed, a 10/xl aliquot of PCR product was 
electrophoresed in 1% agarose gel in EtBr to visualize the PCR 

■■■-'■'- ■ --1 i ' 


DNA Sequencing and Expressing 

The 1.6 kb PCR product was digested and ligated into 
pGEX-2T for expression in E. coli by colleagues in Dr, 
Maclaren's lab. Briefly, 1 /xl of the ligation reaction was 
transformed into competent E. coli DH5a cells (GIBCO BRL, 
Gaithersburg, MD) . The E. coli clones carrying the 1.6 kb 
tyrosinase insert were screened by plasmid mini -prep and 
identified by restriction digestion. Once the correct clone 
was identified, it was grown up again and the recombinant 
plasmid was purified using the QIAGEN Plasmid Maxi Kit 
(QIAGEN, Chatsworth, CA) . The highly purified plasmid was sent 
to LAX Sequencing Technologies Inc and the insert was 
sequenced by Sanger's dideoxy-mediated chain termination 
method. The sequencing result confirmed that the 1.6 kb was 
indeed human tyrosinase. For expression, the E. coli clone 
containing tyrosinase insert was grown in LB (Luria-Bertani) 
medium containing ampicillin overnight at 37°C in a shaking 
incubator. The overnight culture was diluted l/lO and 
incubation was continued for 2 hour. The expression of 
tyrosinase protein was induced by adding Isopropyl J5-D- 
thyogalactoside (IPTG) . 


The amplified tyrosinase cDNA appears as a 1.6 kb band on 
1% agarose gel electrophoresis as expected (Figure 1) . The 
remainder of the amplified DNA was purified by Wizard PCR 
Preps DNA purification system from Promega (Madison, WI) . An 
aliquot of the purified DNA was checked by agarose gel 
electrophoresis and the remaining purified DNA was digested 
and ligated into expression vector and the tyrosinase protein 
expressed as described above. The reactivity of tyrosinase 
with autoantibodies from the sera of patients with vitiligo 
was tested by immunoblotting by colleagues from Dr. Maclaren's 
lab. Among the 26 vitiligo sera tested, 61% of them were 
positive for tyrosinase autoantibodies. 


Although autoantibodies against melanocytes have been 
well documented in the literature, the pathogenic role of 
these autoantibodies remains unknown. Since tyrosinase is an 
intracellular enzyme, autoantibodies could not penetrate the 
cell and reach the enzyme intracellularly . Alternatively, the 
autoimmune attack to melanocytes could have been initiated by 
autoreactive T cells such as CTLs and autoantibodies to 
tyrosinase as well as other intracellular components of the 

Figure 1. PCR amplification of human tyrosinase cDNA. 
EtBr stained agarose gel shows the PCR product amplified from 
human tyrosinase cDNA. The PCR product appears as 1.6 kb as 
indicated by DNA marker. M: DNA marker; T: tyrosinase. 




- 1.605 

i 4 

: 36 ^ / . v,T r=^^ f\ 

cell could be produced as a secondary event. Our present study 
confirmed the autoimmune nature of vitiligo, particularly in 
patients who also suffered from other autoimmune disorders. 

■ f 




Gonadal autoantibodies are found in some patients with 
Addison's disease and hypogonadism. The autoantibodies react 
to steroid hormone producing cells of the adrenal cortex, 
placental syncytiotrophoblast , Leydig areas of testis, and the 
theca interna/granulosa layers of ovarian follicles. ^^^ These 
steroidal cell autoantibodies precede the onset of ovarian 
failure in patients with type I APS. The antigens targeted by 
steroidal cell autoantibodies have been suggested to be the 
combinations of the P450 side chain cleavage and IVot- 
hydroxylase enzymes. ^^^ 

Ant i -oocyte autoantibodies have also been detected in 
infertile women which appear to block the adherence and 
penetration of the sperm through the zona pellucida of the 
Q^^_i23 rpj^g zona pellucida is composed of three major 
glycoproteins: ZPl, ZP2 and ZPS.'^" ZP3 , the primary sperm 
receptor at fertilization, has been identifed as an 
autoantigen in experimental murine autoimmune oophoritis, ^"'^^* 
a model of human premature ovarian failure. Therefore, the 
possibility that ZP3 could be an autoantigen in human gonadal 


failure was investigated. 

Materials and Methods 


I examined sera from 13 patients with gonadal failure. 
All of them were female, some of them had antibodies to 
pituitary, some had antibodies to GAD. I also studied 8 normal 
disease -free controls. None of the normal controls had 
autoantibodies to pituitary, GAD, or thyroid antigens. 

In vitro Translation and Immunoprecipitation 

The cDNA clone of human ZP3 (under T7 promoter) was 
provided by Dr.Jurrien Dean (Laboratory of Cellular and 
Developmental Biology, NIDDK) . The expression of ZP3 was 
examined by the positive control antibody (monkey anti-human 
ZP3) provided by Dr. Kenneth Tung, Dept . of Pathology, Univ. of 

The recombinant plasmid was propagated in E.coli and 
purified by the Magic™ Minipreps System (Promega) . The ZP3 
cDNA was transcribed and translated as described according to 
the manufacturer's instructions (Stratagene, La Jolla, CA) . In 
brief, 1/xg circular plasmid DNA was transcribed in a lOO/zl 

' 1 

reaction for 2 hours at 40°C, using SP6 RNA polymerase in the 
presence of RNAsin. The translation was done using a 
methionine -free rabbit reticulocyte lysate (Promega) in a 50 
/xl reaction using 20% of the synthesized RNA as substrate in 
the presence of 4 [il ^^S-methionine (10 mCi/ml) (Amershara, 
Arlington Heights, IL) . - ^ 

Once the translation reaction was complete, the 
translated products were examined by taking 5 ^1 aliquots 
mixed with 2 fil of SDS sample buffer. The samples were heated 
at 100°C for 3 minutes and subjected to a 10% SDS-PAGE. For 
autoradiography, the gels were dried and exposed to X-ray film 
(XAR-2 ready pack, Sigma, St Louis, MO) overnight at room 
temperature. Since ZP3 is a glycoprotein, canine pancreatic 
microsomes (Promega) were added to the translation reaction 
mixture in order to obtain the mature glycosylated receptor. 
For characterization of the autoantibody reactivities, the 
translated products (50,000 cpm) were incubated at 4°C 
overnight with 2 /xl of sera diluted in PBS with a final volume 
of 100 ^1. The immunocomplexes were washed three times with 
ice-cold PBS in the presence of either 1% or 0.5% Triton X-100 
and incubated with protein A - Sepharose beads for another 45 
minutes. After washing, 50 /zl of the SDS gel loading buffer 
were added to the bead and boiled for 3 minutes. 
Autoradiography were performed as above. 




? ■ , 

The full length ZP3 cDNA was translated as a 45-50 kDa 
double band. The 50 kDa band represents the glycosylated form 
of the protein since it appeared only in the presence of 
microsome membranes, particularly in the presence of 2 ^1 
membranes. Both the glycosylated and non-glycosylated form of 
ZP3 were recognized by the monkey anti-ZP3 antibody (Figure 
2) . None of the patient's sera or normal controls reacted to 
ZP3 (Table 4) . 

These preliminary results indicated that autoantibodies 
against human ZP3 were not detected by this technique. Since 
some autoantibodies may have low affinity to their antigens 
and may dissociate under high concentrations of detergent, the 
immunoprecipitation was also performed using 0.5% Triton X- 
100. However, as shown in Figure 2, similar results were 
obtained as in Figure 3 . 


Experimental autoimmune oophoritis can be induced by 
immunizing mice with peptide derived from ZP3.^" Adoptive 
transfer experiment has shown that CD4* T cells specific for 
ZP3 peptide were able to induce the disease. I hope that 

Figure 2. Immunoprecipitation of human ZP3 . 

The in vitro translated human ZP3 was immunoprecipitated by a 
monkey anti-ZP3 antibody (lane 1) . The 45 and 50 kDa bands 
represent the non-glycosylated band and glycosylated form of 
ZP3 respectively. The serum from normal monkey (lane 2) , 
patients with gonadal failure (lane 3-4) and healthy control 
(lane 5) did not react with ZP3 . 


5 4 3 2 1 

• ■"^''-' 


Table 4. Autoantibody reactivity to ZP3 (Immunoblot) 


Antibody to ZP3 

Patients with APS I 


Patients with APS II 

0/4 1 

Isolated gonadal failure 


Normal control 


Monkey anti-ZP3 serum 


Normal monkey serum 



Figure 3. Immunoprecipitation of human ZP3 with reduced amount 
of detergent . 

The in vitro translated human ZP3 was immunoprecipitated by a 
monkey anti-ZP3 antibody (lane 1) . The 4 5 and 50 kDa bands 
represent non-glycosylated band and glycosylated form of ZP3 
respectively. Reduced amount of detergent (0.5%) was used in 
the washing step. The serum from normal monkey (lane 2) , 
patients with gonadal failure (lane 3-4) and healthy control 
(lane 5) did not react with ZP3 . 

^ - i 


5 4 3 2 1 


• ' *■ • ... 


autoantibodies against ZP3 might also be present in the 

circulation of human patients with hypogonadism. However, ZP3- 

specific autoantibodies have not been demonstrated so far. 

Autoantibodies to zona pellucida were developed by immunizing 

the {C57BL/6 x A/J) Fl (B6AF1) mice with a ZP3-derived peptide 

which is a known T cell epitope/^'' but does not contain a B 

cell epitope. Thus a pure T cell reactive peptide has elicited 

autoantibodies through immunizations against a region of ZP3 

outside the T cell peptide. The authors termed the 

autoantibodies produced in this circumstance "amplified 

autoantibodies". They concluded that the B cells responded to 

endogenous ovarian antigen after activation of ZP3 -specific T 

helper cells, and that the induced autoantibodies do not have 

to mirror the immunogen that initiates the autoimmune process. 

Immunofluorescence studies have confirmed that the 

autoantibodies reacted to zona pellucida. However, the authors 

did not show that the autoantibodies were directed 

specifically against ZP3 as the zona pellucida contains 

numerous other proteins. Other approaches such as 

immunoprecipitation or immunoblot using either biochemically 

purified or recombinant ZP3 should provide direct answers 

concerning the specificity of these autoantibodies. 

Since autoantibodies bind to zona pellucida as confiirmed 

by immunofluorescence, it is possible that ZPl and/or ZP2 may 

also be involved in the autoimmune process. However, only ZP2 

u -r '•' 

and ZP3 were transcribed in growing oocytes and neither of 
them has been detected in resting oocytes. Since ZP3 is the 
primary sperm receptor and ZP2 is secondary sperm receptor, 
Dr. Tung's group has mainly focused on ZP3 in animal 
studies. ^^^ Although there is no overall similarity in the 
amino acid sequences of ZP2 and ZP3, they share two common 
structural motifs at the C- terminal regions. Therefore, the 
possibility that immunization of ZP2 would also induce 
oophoritis in mice should be tested. 

In the present study, I have attempted to demonstrate the 
ZP3 -specific autoantibodies using in vitro translated ZP3 as 
antigen by immunoprecipitation. However, no autoantibodies 
were detected in any of the sera tested. The translated ZP3 
appeared as a 45-50 kDa band as expected and the translated 
product was recognized by the monkey anti-ZP3 antibody. 
Therefore, the failure of detecting autoantibodies was likely 
not due to technical problems. One possible explanation could 
be that if the autoantibodies do exist, they may be present at 
very low titers, below the level of detection by this 
technique. Other approaches such as ELISA are usually useful 
in the detection of autoantibodies as initial screenings once 
an antigen has been very well characterized, purified and 
solubilized. However, ELISA is not a good assay for the 
initial investigation of candidate autoantigens unless the 
antigen is readily available as purified and soluble form. In 


the case of ZP3, the protein was translated in the rabbit 
reticulocyte system and it is only suitable for 
immunoprecipitation because the translated ZP3 is radio- 
labelled but in a mixture of many other proteins. Another 
possibility is that the autoantibodies have very low affinity 
such that they may dissociate quickly upon binding to the 




The Anatomy and Physiology of Parathyroid Gland 

Calcium ions are essential for a wide variety of biologic 
functions. This include vital extracellular processes, such as 
blood clotting, intercellular adhesion, and skeletal 
integrity. This also include intracellular processes, such as 
the regulation of hormonal secretion, cell division, and cell 
motility. ^^® 

The homeostasis of calcium and phosphate concentrations 
in the extracellular fluid is maintained by a finely 
integrated regulation of their absorption from the intestine, 
reabsorption from the glomerular filtrate and mobilization 
from the skeleton. The parathyroid glands are the regulatory 
organs that mediate fine control of ionized calcium levels 
through a direct effect on skeletal in bone. At the same time, 
they activate vitamin D in the diet or following its formation 
in the skin after ultra violet light exposure, such that 
intestinal absorption of calcium/phosphate is indirectly 
promoted. ^^® 



Most people have four parathyroid glands situated close 
to the posterior surface of the lateral lobes of the thyroid 
gland. The combined weight of the four glands is approximately 
120 mg. The parathyroid glands of children weigh approximately 
half this amount. The normal epithelial cells comprising the 
gland are of two types: the chief cells and the oxyphil cells. 
The chief cells are considered to be actively engaged in 
synthesizing hormone and to be the main source of parathyroid 
hormone (PTH) . The oxyphil cells are derived from chief 
cells. ^2« 

PTH is a single chain polypeptide of 84 amino acids, 
produced by two enzymatic cleavages at the amino terminus from 
its preprohormone (115 amino acids) . The direct function of 
PTH is to maintain the homeostasis of circulating ionized 
calcium and phosphate concentrations in the extracellular 
fluid within a very tight range. The synthesis and secretion 
of PTH are regulated by the extracellular calcium ion 
concentration which the parathyroid chief cells are very 
sensitive to. 

The Structure and Function of Ca-SR 

The parathyroid chief cells are equipped with a Ca^* 
sensing mechanism which exhibit an unusual inverse 
relationship between the Ca^" levels and PTH release after 

■ \ 

.. - ^ , . ■■ ' I 

- i 

Stimulation of its calcium sensing receptor (Ca-SR) . The 
regulation of PTH secretion requires that parathyroid cells 
sense the free calcium ion levels in extracellular fluid. The 
recently cloned calcium sensing receptor responds to increased 
levels of extracellular calcium by triggering a phospholipase- 
C (PLC) dependent pathway which in turn induces the 
parathyroid cell to decrease its constituent PTH secretion/^' 
The Ca-SR was cloned by screening a cDNA expression 
library in Xenopus laevis oocytes. ^^° The 5.275 kb cDNA has a 
3.255 kb open reading frame encoding a protein of 1,085 amino 
acids with apparent molecular weight of 120-140 kDa . It 
contains a large extracellular domain of 613 amino acids at 
the amino terminus, a central core of 250 amino acids 
containing seven potential membrane -spanning helices 
characteristic of the G-protein coupled receptor superfamily 
and a intracellular domain of 222 amino acids at the carboxyl 
domain (Figure 4) . The external domain probably serves as the 
actual ionized calcium detector. Sequence analysis shows that 
the Ca-SR contains 9 potential N-linked glycosylation sites. 
The Ca-SR gene for human has been mapped to chromosome 3q2. 
Mutations of the Ca-SR gene have been found to be responsible 
for familial benign hypocalciuric hypercalcemia (FBHH) and 
neonatal severe hyperparathyroidism.^^^ 

Figure 4. Diagram of the strategy for in vitro translation of 


The full length Ca-SR cDNA encodes 1085 amino acid. The 

extracellular domain (1-613) and intracellular domain (580- 

1085) were translated separately by a rabbit reticulocyte 




PTH - secreting cell 




The Detection of Autoantibodies in AH in the Past 

An autoimmune etiology for AH has been suggested because 
of its association with other autoimmune diseases, ^^ and by 
reports of autoantibodies directed against the parathyroid 
tissues in affected individuals. Autoantibodies to the 
parathyroid glands were first reported by Blizzard et al.^^^ 
In that study, 3 8% of 74 patients with autoimmune 
hypoparathyroidism were found positive compared with only 6% 
of 245 healthy control subjects. The results from subsequent 
studies were controversial, since the antibodies often 
appeared to be directed against mitochondrial antigens which 
happen to be increased in parathyroid cells. Autoantibodies 
from the sera of patients with sporadic adult onset 
hypoparathyroidism however have been reported to bind to the 
cell surfaces of human parathyroid cells, resulting in an 
inhibition of PTH secretion.^" In addition, autoantibodies in 
the sera of patients with AH have been reported to be 
cytotoxic for cultured bovine parathyroid cells, by an 
antibody mediated cytotoxicity dependent on complement 
fixation and activation. ^^*'^^^ 

Whereas the above findings do constitute evidences for 
the presence of autoantibodies against parathyroid glands in 
AH, the target autoantigens have not been previously 


Materials and Methods 


I examined sera from 25 patients with AH. Of these, 17 
patients had APS I (all of them had AH, 14 had mucocutaneous 
candidiasis, 10 had Addison's disease, and many had associated 
vitiligo, alopecia, chronic active hepatitis and/or primary 
hypogonadism) . Eight patients had adult-onset 
hypoparathyroidism associated with autoimmune hypothyroidism, 
confirmed by the presence of thyroid microsomal antibody 
and/or thyroglobulin antibody (Table 5) . I also studied sera 
from 10 patients with Addison's disease, 10 with Graves' 
disease, 12 with Hashimoto's thyroiditis, 10 with insulin 
dependent diabetes (IDD) and 8 with vitiligo (none of whom had 
AH), as well as 22 normal disease-free controls. None of the 
normal controls had any endocrine-associated serum 
autoantibodies, such as thyroid microsomal, thyroglobulin 
autoantibodies, or islet cell autoantibodies. 

Antigen Preparation 

The human parathyroid glands were placed on ice in PBS 
with a mixture of protease inhibitors (1, 10-phenanthroline, 
aprotinin, EDTA and benzamidine) . The tissues were homogenized 


Table 5. Characteristics of AH patients 




Age of Onset 



lOF & 7M 

1 yr - 12 yr 

AH in Adult 


8F & OM 

31 yr - 53yr 

AH, Acquired hypoparathyroidism; APS I, Type I autoimmune 
polyglandular syndrome; M, Male; F, Female. 


with a glass tissue grinder and centrifuged at 15,000xg to 
remove cell debris, nuclei and mitochondrial proteins. The 
supernatant was centrifuged again at 100,000xg and the 
resulting supernatant (cytosolic) and pellet (membrane) 
fractions were used as antigen sources in the immunoblot and 
absorption studies. , , .,*'»■":,' 

Plasma membrane preparations from HEK-293 cells 
expressing the Ca-SR, together with membrane preparations from 
wild type HEK-293 cells were kindly provided by Dr. Forrest 
Fuller (NPS Pharmaceutical) and used as antigen sources in the 
immunoblot and absorption studies described below. 

The cDNA clone of human parathyroid secretory protein 

(Chromogranin-A) was kindly provided by Dr. Helman-LJ 

(Molecular Genetics Section, National Cancer Institute) . 

Monoclonal antibody to human chromogranin-A (LK2H10) was 

obtained from Boehringer Mannheim (Indianapolis, IN), rabbit 

anti-human chromogranin-A (A430) was from DAKO Corporation 

(Carpinteria, CA) and monoclonal antibody to human 

chromogranin-A (PHE5) was from Enzo Diagnostics (Farmingdale, 

NY) . The negative control antibody anti-lucif erase was from 

Promega (Madison, WI) . 

Immunoblot ting 

The parathyroid gland extract and the HEK-293 cell 


membrane fractions were solubilized in SDS gel loading buffer 
containing DTT and heated for 3 minutes at 100°C before 
loading. After separation by a 8% SDS-PAGE, the proteins were 
transferred onto Immobilon-P membranes (Millipore, Bedford, 
MA) . The strips of the membrane were cut and incubated with 1% 
BSA in Tris-buffered saline and 0.05% Tween-20 (TEST) to block 
free potential binding sites. Test sera at l/lOO dilutions as 
well as purified IgG of a rabbit anti-Ca-SR antisera and IgG 
of pre-immune rabbit sera were incubated with the antigen- 
containing strips. The strips were then incubated with an 
anti-human or anti-rabbit polyvalent immunoglobulin alkaline 
phosphatase conjugate, and developed with 5-bromo-4-chloro-3- 
indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) 
(Promega, Madison, WI) . 

In vitro Translation and Immunoprecipitation 

The human Ca-SR cDNA was obtained from Dr. Edward Brown 
(Brigham and Women's Hospital) and its extracellular and 
intracellular domains were amplified by PCR. The PCR products 
were positioned downstream of the SP6 promoter on the pcDNAS 
construct. The recombinant plasmid was propagated in E.coli 
and purified by the Magic™ Minipreps System (Promega) . The Ca- 
SR cDNA was transcribed and translated as described according 
to the manufacturer's instructions (Stratagene, La Jolla, CA) . 

59 . \ ; 

In brief, 1/xg circular plasmid DNA was transcribed in a 100^1 
reaction for 2 hours at 40°C, using SP6 RNA polymerase in the 
presence of RNAsin. The translation was done using a 
methionine -free rabbit reticulocyte lysate (Promega) in a 50 
111 reaction using 20% of the synthesized RNA as a substrate in 
the presence of 4 ^1 ^^S-methionine (10 mCi/ml) (Amersham, 
Arlington Heights, IL) . 

Once the translation reaction was complete, the 
translated products were examined by taking 5 yil aliquots 
mixed with 20 ill of SDS sample buffer. The samples were heated 
at 100°C for 3 minutes and subjected to 10% SDS-PAGE. For 
autoradiography, the gels were dried and exposed to X-ray film 
(XAR-2 ready pack, Sigma, St Louis, MO) overnight at room 
temperature. Since Ca-SR is a glycoprotein, canine pancreatic 
microsomes (Promega) were added to the translation reaction 
mixture in order to obtain the mature glycosylated receptor. 

For characterization of the autoantibody reactivities, 
the translated products (50,000 cpm) were incubated at 4°C 
overnight with 2 /il of sera diluted in PBS with a final volume 
of 100 111. The immunocomplexes were washed three times with 
ice-cold PBS and incubated with protein A - Sepharose beads 
for another 45 minutes. After washing, 50 ill of the SDS gel 
loading buffer were added to the beads and boiled for 3 
minutes. Autoradiography were performed as above. 


Absorption of Autoantibodies with Recombinant Ca-SR 

The patient sera (2 /xl) were incubated for 2 hours at 
room temperature with 1 mg recombinant Ca-SR expressed by HEK- 
293 cell diluted in 50 /xl PBS. This mixture was then 
centrifuged (13,000g, 15 min) , and the supernatant was again 
subjected to immunoprecipitation with the in vitro translated 
extracellular domain of the Ca-SR as an antigen source, to 
learn whether the positive band (see later) had been removed. 

Results ..' 

The Identification of Autoantibodies in AH 

Autoantibodies against specific proteins were detected in 
both cytosolic and membrane fractions of the parathyroid 

(1)' Autoantibodies to the cytosolic fraction of 
parathyroid gland. 

Seventeen AH patient's sera were tested for their 
reactivity to the cytosolic fraction using immunoblot (Figure 
5) . Twelve (11 had APS I, 1 had adult onset AH) patient sera 
reacted to a protein of 70 kDa, and 16 (15 had APS I, 1 had 
adult onset AH) to one of 80 kDa. 

Sera from 50 patients with other autoimmune diseases as 

Figure 5. Immunoblot analysis using the cytosolic fraction of 
human parathyroid gland extract . 

The cytosolic fraction of the parathyroid gland extract was 
solubilized and separated by a 10% SDS-PAGE. After separation, 
the proteins were transferred onto Immobilon-P membranes. 
Immobilon - P strips containing the parathyroid extract were 
incubated with AH sera (lanes 1-10) and control sera (lanes 
11-12) . The reactivities of the autoantibodies were visualized 
by an alkaline phosphatase mediated BCIP/NBT system. The 
autoantibodies from AH sera reacted to specific antigens with 
molecular weight of 70 kDa (lanes 1,2,3,4,5,6,8) and 80 kDa 
(lanes 1-9). Two patients's sera also reacted to a 60 kDa 
antigen (lanes 4 and 5) . Only one of the AH patients (lane 10) 
was negative for the specific antigens. 


200 kDa 

80 kPa 
70 kDa 

1234 5 678 9 10 11 12 M 

f V 


well as 13 normal controls were also tested, and none of them 
reacted with any of the above specific parathyroid proteins 
(Fig. 6) . 

Since human parathyroid secretory protein (PSP) also 
known as chromagranin A has a molecular weight which is 
similar to the 70 kDa antigen, I investigated the possibility 
that the PSP is a potential antigen. The PSP cDNA was 
translated in vitro under the control of the SP6 promoter. The 
translated protein has a molecular weight of 68 kDa and it 
appears as a non-glycosylated protein (Figure 7) . 

The translated PSP was verified by positive control 
antibodies raised against this protein as shown in Figure 8 , 
Monoclonal antibody PHE5 only shows a faint band for PSP, 
whereas monoclonal antibody LK2H10 and rabbit antibody A43 
show clear staining of the 68 kDa PSP protein. A430 also 
reacted to the 60 kDa protein which might be a truncated form 
of the PSP. The negative control antibody (anti-lucif erase) 
did not react to PSP. Next, the reactivity of the sera from 
patients with AH to PSP was tested. As shown in Figure 9. None 
of patients sera reacted to PSP. 

(2) Autoantibodies to the membrane fraction of 
parathyroid gland. 

Autoantibodies were detected against the membrane 
fraction of parathyroid gland in 5 of 25 (20%) of the AH sera 
by immunoblot. Two patients had APS I and 3 had adult onset 


Figure 6. Immunoblot analysis of the cytosolic fraction of 
human parathyroid gland extract using normal human sera. 
The cytosolic fraction of the parathyroid gland extract was 
solubilized and separated by a 10% SDS-PAGE. After separation, 
the proteins were transferred onto Immobilon-P membranes. 
Immobilon - P strips containing the parathyroid extract were 
incubated with normal human sera (lanes 1-13) . The 
reactivities of the autoantibodies were visualized by an 
alkaline phosphatase mediated BCIP/NBT system. The normal 
human sera reacted to a non-specific band with molecular 
weight of 90 kDa. 


1 2 3 4 5 6 7 8 9 10 11 12 13 

Figure 7. In vitro translation of human parathyroid secretory 
protein (PSP) . 

The in vitro translated products were separated by a 12% SDS- 
PAGE and then visualized by autoradiography. The human PSP 
cDNA was translated as a 68 kDa protein and a minor band of 60 
kDa. Addition of microsomal membranes (lanes 2-3) did not 
cause additional bands indicating that the PSP is not a 
glycoprotein . 




Figure 8. Immunoprecipitation of human parathyroid secretory 
protein (PSP) by control sera. 

The in vitro translated PSP was incubated with monoclonal 
antibodies against human PSP (lane 1: PHE5, lane 2: LK2H10) , 
rabbit anti-human PSP (lane 3: A430) and negative control 
antibody (lane 4: rabbit anti-luciferase) . 

. V 

T t, ,•; it. ^ f , 

, r ' '-^ -' » 




Figure 9. Immunoprecipitation of human parathyroid secretory 
protein (PSP) by sera from patients with hypoparathyroidism. 
The in vitro translated human PSP was immunoprecipitated by 
patient's sera (lanes 1-6), normal human sera (lane 7) and 
positive control sera LK2H10 (lane 8) . Only the positive 
control sera reacted to PSP. " • '* 



AH. The autoantibodies reacted with a doublet 120-140 kDa 
protein in the parathyroid gland extract (Figure 10) . Sera 
from 50 patients with other autoimmune diseases as well as 22 
normal controls were also tested, and none of them was 

The Characterization of Ca-SR in AH 

Since the parathyroid 120-140 KDa antigen has the same 
molecular weight as the Ca-SR dependent upon its degree of 
glycosylation, I tested the possibility that the receptor 
itself was the autoantigen by three different experimental 
approaches . 

In the first approach, the AH sera were tested by 
immunoblot using a membrane fraction of HEK-293 cells 
transfected with Ca-SR cDNA. The patient sera reacted to a 
120-140 kDa protein (Figure 11, lane 2) , which closely matched 
that recognized by the anti-Ca-SR IgG raised in rabbit (Figure 
11, lane 3) . The patient sera at 1:100 dilution did not react 
with other bands. Eight of 25 AH patient sera (32%, 3 APS I 
and 5 adult onset AH) including the above-mentioned 5 positive 
sera reacted to the Ca-SR from this source, but none of the * 
control sera did so. In addition, the 8 positive AH patient 
sera did not react to non-transfected or wild type HEK-293 
cells which did not express Ca-SR proteins (Table 6) . 

Figure 10. Immunoblot analysis using the membrane fraction of 
human parathyroid gland extract . 

The membrane fraction of the parathyroid gland extract was 
solubilized and separated by a 8% SDS-PAGE. After separation, 
the proteins were transferred onto Immobilon-P membranes. 
Immobilon - P strips containing the parathyroid extract were 
incubated with normal sera (lane 1) and AH sera (lane 2) . The 
reactivities of the autoantibodies were visualized by an 
alkaline phosphatase mediated BCIP/NBT system. 






- -•■'■- 






Figure 11. Immunoblot analysis using membranes of HEK-293 
cells transfected with human Ca-SR cDNA. 

The HEK-293 cell membranes were solubilized and separated by 
8% SDS-PAGE, then transferred onto Immobilon-P membranes. 
Immobilon - P strips containing the antigen were incubated 
with normal sera (lane 1) , AH sera (lane 2) , rabbit ant i -Ca-SR 
IgG (lane 3) and pre -immune rabbit IgG (lane 4) . The 
reactivities of the antibodies were visualized by an alkaline 
phosphatase mediated BCIP/NBT system. 



Table 6. Autoantibody reactivity to recombinant 
Ca^*- Sensing receptor (Immunoblot) . 

Ag source 



Ca-SR IgG 

HEK-293 cell 




Wild type 
HEK-293 cell 




AH: Acquired hypoparathyroidism, 


In the second approach, the Ca-SR was translated in vitro 
into two parts in order to identify the antigenic epitopes 
reactive to the putative autoantibody that we had discovered. 
Overlapping extracellular (residues 1-613) and intracellular 
(residues 580-1085) domains of the Ca-SR were expressed as 
shown in Figure 4. The extracellular domain was translated as 
shown in Figure 12. Two bands with the molecular weight of 46, 
and 60 kDa are seen in lane 1. Glycosylation occurs with the 
addition of canine pancreatic microsome membranes. As can be 
seen in lanes 2-5, this step induced one additional band to 
appear as 70 kDa, meanwhile, the intensity of the 60 kDa band 
decreased by 50% as expected for glycosylated proteins. 
Apparently, the 60 kDa is the non-glycosylated form and 70 kDa 
is the glycosylated form of extracellular Ca-SR. The 46 kDa 
band, however was unexpected. To determine whether it was a 
form of Ca-SR or an unrelated artifact of the translation 
system, I immunoprecipitated the translated extracellular 
domain by the rabbit anti-Ca-SR antibody. This rabbit antibody 
was raised against a peptide of the extracellular domain. As 
shown in Figure 13, the antibody recognized all three bands, 
suggesting that the 46 kDa band is also a portion of the Ca- 
SR, perhaps a degraded or truncated product. 

The patient sera reacted to 60 and 70 kDa forms of the 
extracellular domain but not the 46 kDa band (Figure 14) 
indicating that the autoantibodies recognized different 

Figure 12. In vitro translation of the extracellular domain of 
the Ca-SR. 

The in vitro translated products were separated by a 10% SDS- 
PAGE and then visualized by autoradiography. The extracellular 
domain of the Ca-SR was translated as 46 and 60 kDa protein 
bands (lane 1) . One additional band (70 kDa) appears when 1-4 
/il of microsomal membranes were added to the reaction (lanes 
2-5) . 





..« ' 

•I «.< 

Figure 13. Immunoprecipitation of the extracellular domain by 
rabbit anti-Ca-SR. 

The in vitro translated extracellular domain of the Ca-SR was 
incubated with a rabbit anti-Ca-SR IgG (lane 1) or with a pre- 
immune rabbit IgG (lane 2) . Samples were precipitated by 
protein-A-Sepharose, separated by a 10% SDS-PAGE and then 
visualized by autoradiography. 






Figure 14. Immunoprecipitation of the extracellular domain by 
AH sera. 

The in vitro translated extracellular domain of the Ca-SR was 
incubated with AH sera (lanes 1-3) or with normal control sera 
(lanes 4-6) . Samples were precipitated by protein-A-Sepharose, 
separated by 10% SDS-PAGE and then visualized by 
autoradiography . 

H»*. - . t'- 



1 2 3 4 5 6 

■'■-' ■■*,.. V-- - •.■»^ 


85 ■■ ■ ■•■" ' ' ■ "■ 
epitopes compared to the rabbit antibody. By using this 
technique, 14 of 25 (56%) AH sera were positive. Six had APS 
I and 8 had adult onset AH. Furthermore, glycosylation is not 
required in the formation of all autoantibody reactive 
antigenic epitopes since both the non-glycosylated (60 kDa) 
and glycosylated (70 kDa) proteins were recognized. None of 
the control sera reacted to the extracellular domain of the 

In the third approach, the positive AH sera were pre- 
incubated with the HEK-293 membranes containing the Ca-SR. The 
reactivity of the sera was completely removed after the pre- 
absorption. As shown in Figure 15, the AH sera reacted with a 
60 and 70 kDa protein before absorption (lane 1) and the 
reactivity disappeared after the absorption (lane 2) . 

The cytosolic domain was translated as a 60 kDa protein 
and no glycosylation occurred after exposure to the microsomal 
membranes as expected. None of the patient sera reacted with 
the cytosolic or intracellular domain of the Ca-SR (Table 7) . 

In summary, 14 (56%) of AH patient sera reacted to the 
extracellular domain of the recombinantly expressed Ca-SR, 
whereas none of the 25 AH patient sera reacted to the 
intracellular domain of the molecule. The 14 antibody positive 
patients which responded to the extracellular domain of the 
Ca-SR included all 8 positive patients that had reacted to the 
transfected HEK- 293 cells. The autoantibody frequencies might 

Figure 15. Absorption studies with HEK-293 cells. 
The extracellular domain of the Ca-SR was immunoprecipitated 
by a positive AH sera before (lane 1) and after (lane 2) pre- 
absorption with HEK-293 cell membranes containing the Ca-SR. 
Samples were precipitated by protein-A-Sepharose, separated by 
a 10% SDS-PAGE and then visualized by autoradiography. 





1 2 


Table 7. Autoantibody reactivity to in vitro translated 
domains of calcium sensing receptor 
(immunoprecipitation) . 







14/25 (56%) 


Addison's disease 



Graves' disease 











0/8 1 

Normal control 



Ca-SR, calcium sensing receptor; IDD, insulin dependent 
diabetes; AH, acquired hypoparathyroidism. 


have been higher if newly diagnosed patients had been 
exclusively studied. None of the 22 normal control sera 
reacted to either domain of the Ca-SR. Sera from 50 patients 
with other autoimmune diseases were also tested, and none of 
them reacted to either domain of the Ca-SR (Table 7) . 


Our studies confirm the autoimmune nature of AH and 
demonstrate that autoantibodies in patients with AH target 
human parathyroid proteins of 70, 80 and 120-140 kDa . The 
autoantigens are disease specific since they were only 
recognized by the sera from patients with AH and not from 
those with other autoimmune diseases. 

The autoantigens are not mitochondrial proteins, because 
the mitochondrial proteins were removed by centrifugation 
prior to immunoblotting in our study. Furthermore, we chose 
fresh human hypercellular parathyroid glands and normal dog 
parathyroid glands instead of parathyroid cell lines or 
tumors, so that we could avoid spurious identification of any 
antigens that are not normally present in the parathyroid 

Most of the Ca-SR autoantibody positive patients 

(including 5 AH in the context of APS I and all 8 adult-onset 

AH in association with thyroid disease) were females (Table 



8) . This finding of female predominance is consistent with 
results in other autoantibody mediated diseases targeted at 
membrane receptors. Four of our adult -onset AH patients 
developed their disease and had the Ca-SR autoantibodies 
detected after they had babies, another 2 adult -onset AH 
patients developed their disease after menopause, while one 
who presented with AH in the context of APS I began her 
disease at the onset of her menses. These findings suggest a 
possible influence of female hormones in the manifestation of 
the disease, as in many other autoimmune syndromes. 

That autoantibodies to Ca-SR were absent from some AH in 
the context of APS I could be possibly explained by the 
complete loss of the autoantigen needed to drive their 
formation long before we could study them. Two of the Ca-SR 
autoantibody negative AH patients had developed their disease 
32 years ago, while another 2 autoantibody negative AH 
patients had their diseases for more than 10 years at the time 
of this study. However, we were able to collect a serum sample 
immediately after the onset of AH from a 34 years old female 
who developed AH after an infection by influenza and she 
happened to be strongly positive for Ca-SR antibody. A general 
characteristic of all autoimmune diseases is that there are 
remissions and exacerbations of the underlying pathogenic 
processes involved over time. With IDD, islet cell 
autoantibodies (ICA) disappear following clinical onset of 


Table 8. Characteristics of positive AH patients. 




in vitro 

AH in APS I 


lOF & 7M 

5F Sc IM 

AH in Adult 


8F & OM 

8F & OM 

AH, Acquired hypoparathyroidism; APS I, Type I autoimmune 
polyglandular syndrome; M, Male; F, Female. 


disease when the pancreatic S cells are destroyed, and the ICA 
reactive self antigens have disappeared. In some diabetic 
patients, even with the combination of different well defined 
antigens, autoantibodies are never detected. AH may have a 
similar course in respect to the Ca-SR autoantibody. 
Alternatively, different antigens may exist in different 
patients or it is possible that in some patients they simply 
do not appear at any time. Finally, the Western blot technique 
I used, although specific for antigen reactivity, is a 
relatively insensitive method for detecting autoantibodies. 
Other more sensitive assays, such as radioimmunoassay or 
ELISA, may be able to increase the autoantibody frequencies.^" 
This possibility remains to be explored. 

The Ca-SR appeared as a 120-140 kDa bands on immunoblot 
and the external domain of Ca-SR appeared as a 60-70 kDa bands 
on immunoprecipitation due to differential glycosylation of 
the receptor components. However, this differential 
glycosylation did not appear to affect the antigenic structure 
of the Ca-SR since both the 120-140 kDa and 60-70 kDa bands 
were well recognized by both the rabbit antibody and the AH 
patient sera. The transfected HEK-293 cells contain more Ca- 
SRs than the normal human parathyroid gland membrane 
preparations and this may explain why more of the AH patients 
were found to be positive when the HEK-293 membranes rather 
than human parathyroid gland membranes were used as antigen 


sources in immunoblotting (Table 9) . In addition, the in vitro 
translated external domain of Ca-SR had much less background 
than the transfected HEK-293 cells and this may explain why as 
many as 56% of the AH patients were found to be positive when 
in vitro translated external domain of Ca-SR was used as the i 
antigen source in immunoprecipitation studies. 

I have yet to identify the 70 and 80 kDa parathyroid 
protein antigens. Parathyroid secretory protein (PSP) has a 
molecular weight of 70 kDa and is co- stored and co- secreted 
with PTH in the parathyroid gland. ^^^ The protein has been 
shown to be present in the secretory granules of a variety of 
neuroendocrine and endocrine tissues with greatest abundance 
in parathyroid glands and adrenal medulla. I therefore tested 
the reactivities of the positive sera with PSP using the in 
vitro translation based immunoprecipitation technique, but 
found no positive reactivities. 

Many membrane proteins and serum proteins contain 
carbohydrate chains called glycoproteins, which often 
contribute importantly to the folding and stability of the 
proteins as well as to their synthesis and positioning within 
a cell, especially as integral membrane proteins. Sugar 
residues in glycoproteins are commonly linked to two different 
classes of amino acid residues. The sugars are classified as 
0- linked if they are bonded to the hydroxyl oxygen of serine, 
threonine, and (in collagen) hydroxy lysine; whereas they are 


Table 9. Autoantibody reactivity to Ca-SR, 


Antigen source 



In vitro 

AH in APS I 




AH of adult 









classified as N- linked if they are bonded to the amide 
nitrogen of asparagine. It is possible that the 70 and 80 kDa 
autoantigens represent the same molecule with differential 
glycosylation. To test this hypothesis, the cytosolic 
fractions of the parathyroid gland have been incubated with 
endoglycosidase H and 0-glycosidase which cleave N- and 0- 
linked oligosaccharide respectively. After incubation, the 
enzyme treated cytosolic fraction was used as antigen source 
for immunoblot. The 70 and 80 kDa antigen were found unchanged 
after this treatment indicating that they are not 
glycoprotein. This would be consistent with their cytosolic 

There are still other approaches to identify the nature 
of the 70 and 80 kDa antigen. One of the approaches would be 
to purify and sequence the protein and design a DNA probe 
according to the partial amino acid sequence of the proteins 
and then use the probe to screen a cDNA library. The cytosolic 
fraction was separated by SDS-PAGE and stained with Commasie 
brilliant blue. The 70 and 80 kDa region were crowded by 
multiple minor bands. No dominant band in sufficient quantity 
could be isolated for sequencing purpose. This result 
indicated that the 70 and 80 kDa protein may not have a 
dominant function unique to the parathyroid cells. 

The role of Ca-SR autoantibodies in the pathogenesis of AH 
is not known. The positive patient sera were sent to Dr. 

■--'■' »€ - 

Fuller's group to test this possibility on the HEK-293 cells 
transfected with Ca-SR. This established transfected cell line 
expresses Ca-SR on the plasma membrane and responds to 
extracellular calcium by increasing the intracellular Ca^* 
level. Our positive sera did not show any effect on the 
intracellular Ca^* level in this cell line. This result 
indicates that the autoantibody against Ca-SR may not have an 
functional effect, instead, it may fix complement to lyse the 
cell or just be an indicator of the autoimmune process. The 
pathogenic event might also involve cytotoxic lymphocytes 
rather than autoantibodies. The specificity of our results 
however argue best for a possible role of autoimmunity to the 
Ca-SR in AH. 

Our detection of autoantibodies to the Ca-SR in AH could 
lead to the development of a diagnostic test for the disease, 
as well as possibly provide antigen mediated immunotherapies 
based upon the use of recombinant protein antigen as a 
therapeutic agent to restore immune tolerance in AH. 

f . ^r f' 

■ - . ? 



^•- »' 

.. .- f 

>. % H^t'. / ' * 





Alopecia areata is a common condition that results in the 
loss of hair on the scalp and elsewhere. Potentially- 
reversible, it is characterized by either limited patchy hair 
loss (alopecia areata, patchy AA) , loss of all scalp hair 
(alopecia totalis, AT) , or loss of all body hair (alopecia 
universalis, AU) .^^^ 

Alopecia occurs in males and females of all ages, but 
young persons are affected most often. The incidence of 
alopecia areata has been reported at about 17 per 100,000 per 
year, therefore approximately 1% of the population will have 
been affected by the age of 50.^^^ 

The etiology of alopecia areata is not clear. Occasional 
unexplained outbreaks of alopecia areata in closed communities 
have been reported and it has been postulated that viral 
infection might serve as a trigger mechanism for an imbalance 
in a T-cell subpopulation provoking onset of alopecia areata. 
It is known that stress can profoundly affect the immune 
system and many physicians feel that psychological factors may 
play a part in alopecia areata. 



AA has been suggested to be an autoimmune disease because 
of its association with other autoimmune disorders, ^^''^*° an 
inflammatory infiltrate of activated T cells surrounding the 
hair follicles (HFs) in affected areas, and deposits of 
immunoglobulin and complement around HFs particularly at the 
edge of active lesions. Recent studies have shown that serum 
from patients with alopecia areata significantly inhibited the 
growth of normal dermal papilla cells, implicating a serum 
factor reacting to dermal papilla cells. ^^^ 

Cortisone injection and the application of anthralin 
cream are being used for mild patchy alopecia areata. Topical 
immunotherapy is being used for extensive alopecia areata, or 
alopecia totalis/alopecia universalis. Chemicals such as 
dinitrochlorobenzene (DNCB) or diphencyprone (DPCP) are 
applied to the scalp to produce an allergic rash which 
resembles poison oak or ivy. Approximately 40% of patients 
treated by this way will regrow scalp hair. The mechanism for 
this treatment is not known. Recent studies from Happle et 
al"^ has indicated that the beneficial effect of DPCP may be 
mediated by cytokines locally released during the contact 
allergy. They perfomed a semiquantitative RT-PCR with RNA 
extracted from scalp biopsies that were obtained from patients 
with AA before and after successful treatment with DPCP. They 
found an increased mRNA levels for IL-2, IL-8, IL-10 and TNF- 
a. The overall expression level for IFN-gamma was reduced by 


60% compared to untreated AA. These studies provided 
experimental evidence that cytokines may participate in the 
pathogenesis of AA and that T cells might trigger the hair 
loss by releasing IFN-gamma. 

Autoantibodies against normal human hair bulbs have been 
recently demonstrated by Dr. Maclaren's group using indirect 
immunof luorecense technique. Following this finding, I started 
to investigate the potential alopecia autoantigens using 
immunoblot and immunoprecipitation techniques. 

Materials and Methods 

Antigen Preparation ' • ' ' '' = 

A portion of the same crown head human skin as used in 
immunofluorescence was homogenized on ice in the presence of 
1% Nonidet P-40 and protease inhibitors (1 mM 
phenylmethylsulfonyl fluoride, 10 mM aprotinin, 10 mM 
leupeptin, 10 mM iodoacet amide) . After homogenization, the 
homogenate was incubated on ice for 3 mins . The total 
homogenate was used directly for immunoblotting. In addition, 
the skin homogenates were centrifuged at 13,000 g for 30 min 
at 4°C and the supernatant was used for immunoprecipitation. 


The skin homogenates were boiled in SDS- loading buffer 
and loaded to a 12% SDS-PAGE. The separated proteins were 
transferred onto Immunobilon-P membrane by a semi-dry 
electrotransfer unit. Immobilon-P strips were incubated with 
1% BSA in Tris-buffered saline and 0.05% Tween-20 to block 
free binding sites. Test sera at l/lOO dilutions were 
incubated with the antigen-containing strips. The strips were 
then incubated with an ant i -human polyvalent immunoglobulin 
alkaline phosphatase conjugate, and the indicator colour 
developed with 5-bromo-4-chloro-3-indolyl phophate (BCIP) and 
nitro blue tetrazolium (NBT) . 


The two most positive patient sera, together with two 
normal controls were initially analyzed. The skin extract was 
quantitated by Bradford's method, and 200 fjig of protein were 
loaded on the gel. As can be seen from Figure 16, both 
control (lanes 1-2) and patient sera (lanes 3-4) reacted to a 
protein of 60 kDa. The 60 kDa protein thus appeared to be a 
non-specific antigen for the disease. However, the patient 
sera also reacted specifically to additional proteins. Both 
patient sera (lanes 3-4) reacted to three identical proteins 

Figure 16. Immunoblot analysis using human head skin 
homogenate . 

Aliquot of the total human head skin homogenate was 
solubilized and separated by a 12% SDS-PAGE. After separation, 
the proteins were transferred onto Immobilon-P membranes. 
Immobilon - P strips containing the head skin extract were 
incubated with control sera (lanes 1-2) and alopecia sera 
(lanes 3-4) . The reactivities of the autoantibodies were 
visualized by an alkaline phosphatase mediated BCIP/NBT 





12 3 4 


/"» '-c 

with molecular weights between 46 to 55 kDa. These two 
positive sera also reacted to different proteins in addition 
to the common ones. One of them (lane 3) reacted to a protein 
of 57 kDa, the other serum (lane 4) reacted to several 
proteins of high molecular weight (above 100 kDa) . 

In addition to the above-mentioned two patient sera, I 
also tested 9 other AA sera by immunoblot . Four of the 9 sera 
were positive by this method. All of the four positive sera 
reacted to 46 - 55 kDa antigens (Table 10) . Three of the 11 AA 
sera also reacted to the high molecular weight proteins (>100 
kDa) . 

There is a good correlation between the immunoblot and 
immunofluoresence findings. Sera that stain all layers of the 
hair follicle on immunofluoresence reacted to both high and 
low molecular weight antigens. Those that stain only the outer 
layer of HF reacted only to the low molecular weight antigens 
(46 - 55 kDa) . These results indicate that the antigens may be 
located in different regions of the HFs or different types of 
cells may be attacked in the autoimmune process. 


Autoantibodies directed to human hair follicles (HFs) 
have been demonstrated recently by Tobin and Bystryn et al."^ 
Autoantibodies reacted to specific autoantigens derived from 


Table 10. Autoantibody reactivity to human head skin 
homogenate . 


Patients with alopecia 

46 - 55 kDa 


>100 kDa 

3/11 1 

57 kDa 

1/11 1 

'-: . j-- I > 

keratinocytes and melanocytes derived from HFs but not from 
the same type of cells from epidermis. In their immunoblot 
assay, the autoantibodies to HF keratinocytes reacted to 
antigens with molecular weight of 48-50, 52-54, 58-60, and 62- 
64 kDa. The same research group has also developed a mouse 
model for alopecia. The C3H/HeJ mice develop alopecia with age 
that quite closely resembles human alopecia. The mice 
autoantibodies reacted to antigens of approximately 46, 50, 
55, 60 and/or 64 kDa. The autoantigens identified so far by 
this group matched our findings, at least in respect to the 
46-55 and 57 kDa antigens. Most interestingly, autoantibodies 
to some of these antigens in the mice were detected in the 
littermates who had not yet developed hair loss. The authors 
concluded that the autoantibodies may be a cause rather than 
a result of hair loss. An autoantibody transfer experiment 
will be needed to establish the causal role of the 

According to the literatures, no proteins which have 
similar molecular weights to these autoantigens and are unique 
for melanocyte or keratinocyte have been found so far. 
Therefore, I am not able to speculate a candidate autoantigen 
at this moment. 

The nature of these autoantigens will be identified by a 
different approach. My next step is to get partial amino acid 
sequence of the above-mentioned antigens. DNA probes will be 

designed based on the knowledge of the amino acid sequence, 
and the probes will be used to screen a cDNA library generated 
from human skin mRNAs . 


Since the initial demonstration of organ specific 
autoantibodies by Doniach and Roitt in 1957, autoimmune 
processes involved in endocrine diseases have been extensively- 
investigated. With the rapid development of recombinant DNA 
technology, particularly during the last decade, many of the 
major autoantigens involved in autoimmune endocrine diseases 
have been identified as summarized in Table 1. Studies on the 
correlation between the autoantibodies and the disease 
processes have been fruitful in terms of prediction and 
prevention of some of the diseases. While much attention has 
been focused on the major endocrine diseases such IDD and 
autoimmune thyroid diseases, less information is available 
about type I APS . The topic of the autoimmune polyglandular 
syndromes has long been of interest to both endocrinologists 
and immunologists . Collectively, these diseases are very 
common. My thesis has focused on the identification of 
autoantigens involved in hypoparathyroidism and associated 
diseases such as vitiligo, gonadal failure and alopecia 
areata, all of which are component diseases of type I APS. 

A scientific issue that needed to be resolved was whether 
multiple glandular autoimmunities affected individual patients 


and their families because they made autoimmune responses to 
a single antigen commonly present in multiple tissues. This 
possibility appears unlikely, as exemplified by our findings 
as well as those of others. The autoantigens all seem 
different for the component diseases as summarized in Table 1. 
One exception is IVo;- hydroxylase and P450scc which are found 
in steroid cells of adrenal cortex, testes and ovary. Organ 
specific autoantigens may also share common motifs with each 
other or with invading microorganisms. In fact, 21 -hydroxylase 
and 17q;- hydroxylase share a common motif around the steroid 
binding site, however, autoantibodies from patients with 
Addison's disease reacted only with the 2 I -hydroxylase . 
Autoimmune reactivity to common motifs of unrelated proteins 
has been demonstrated at T cell level. A nonamer peptide from 
murine nicotinic acetylcholine receptor which shares four 
amino acid residues with a nonamer peptide of murine ZP3 was 
able to induce murine autoimmune oophoritis.^^* 

Knowledge of the nature of the autoantigens involved in 
APS now allows us to speculate about the dominant pathogenic 
events involved. In the case of type I APS, a Th2 like 
antibody mediated process may be operating as documented by my 
recent identification of the external domain of the calcium- 
sensing receptor as a major autoantigen in hypoparathyroidism. 
In addition, researchers from two different groups have shown 
that IgG from patients with type I APS and Addison's disease 

inhibited ACTH-stimulated Cortisol secretion by guinea pig 
adrenal cells. Although direct immunological evidence (e.g., 
immunoprecipitation) is lacking, it is logical to think that 
the targeted autoantigen in this case may be the ACTH 
receptor. Anti-FSH receptor antibodies have also been reported 
in the literature. On the other hand, type II APS appears to 
be mediated by autoreactive CD8* cytotoxic T cells (a Thl 
like cellular immune response) . The component diseases with 
evidence for this include IDD, Hashimoto's thyroiditis and 
vitiligo. Graves' disease has long been considered to be 
mediated by TSHR-Ab, however, the role of autoantibodies in 
Graves' disease has been questioned since they show only poor 
correlations with the disease process and it is now believed 
that autoreactive T cells contribute to the pathogenesis of 
the disease. Addison's disease may be mediated by a Thl like 
response in the context of type II APS, whereas a Th2 like 
response may be responsive if the disease occurs in the 
context of type I APS. ; ; 

If anti-Ca-SR autoantibody is pathogenic, its mechanism 
of disease initiation is unknown. Autoantibody to 
acetylcholine receptor (AChR) is a good example of pathogenic 
autoantibody. This autoantibody is present in most patient 
with myasthenia gravis (MG) . Although a significant number 
(>10%) of MG patients including some with severe generalized 
weakness have no detectable autoantibodies to AChR, the 

• ■ :'■■■■;>' 

'".,. ■ 110 V " ' ».* r r^ 

pathogenic role of this antibody have been well established. 
Elimination of the autoantibody by immunosuppression therapy 
resulted in marked improvement in the majority of MG patients. 
It is known that the AChR is lost at the motor end-plates due 
to the autoantibodies. The exact way that the autoantibodies 
exert their effect is not known. The antibody may cross-link 
AchR and increase the rate of receptor degradation or antibody 
may fix complement. 

Although autoantibodies may not be pathogenic in type II 
APS, the detection of the autoantibodies may provide valuable 
marker tools for clinical diagnosis and possible prevention of 
the diseases. The identification of the reactive autoantigens 
has made this possible. In IDD, beta cells of the pancreas are 
destroyed as the result of an autoreactive T cell attack. 
Autoantibodies to islet cell antigen may be produced as a 
secondary event which may occur several years before the onset 
of diabetes. The detection of these autoantibodies has proven 
to be very valuable in identifying prediabetic patients and 
therefore permitting the design of immunological intervention 
at an early stage . 

In summary, hypoparathyroidism is a major component 
disease of autoimmune polyglandular syndromes. The autoimmune 
nature of hypoparathyroidism has been confirmed by the 
detection of specific autoantibodies and the identification of 
the calcium sensing receptor as an autoantigen is the first 

step toward the understanding of the autoimmune process of the 
disease. Alopecia is a non-endocrine disease associated with 
type I APS. The presence of autoantibodies has been well 
documented and the nature of the involved autoantigens is 
being resolved. 

;; "rrtnr 'P 


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Yangxin Li was born in Changchun, China, on June 6, 1965. 
Yangxin's dream since she was a middle school student was to 
become a scientist like Madame Curie. She won the second award 
in a national mathematics competition when she was 15 years 
old. Yangxin was listed among the top 10 students in Ji-Lin 
province in the national examination for graduated high school 
students. In 1989, she received a Bachelor of Science degree 
in physical chemistry at the University of Science and 
Technology of China (sponsored by the Chinese Academy of 
Science) which recruits top students from all parts of China. 
She then continued to do research at this university. In 1991, 
she went to Sweden to pursue a Ph.D degree where she married 
Yao-hua Song. She came to America with her husband on November 
25, 1991. In 1992, she entered the graduate program at the 
Department of Pathology and Laboratory Medicine, University of 
Florida. After completion of her doctoral program, Yangxin 
will continue research in the field of autoimmune diseases, 
particularly, alopecia areata. , - * 4\J. l , ■•" • • *. ; 


I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 


Noel K. Maclaren, chair 
Professor of Pathology and 
Laboratory Medicine 

I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 

Nancy Dendlow 
Associate Scientist of 

Biochemistry and Molecular 


I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 

< Catherine Hammett -Stabler 
Assistant Professor of 
Pathology and Laboratory 

I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 

Jdel Schif fenbaner 
Associate Professor of 

Molecular Genetics and 


I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 

Jin-Xiong She 
Assistant Professor of 

Pathology and Laboratory 


This dissertation was submitted to the Graduate Faculty 
of the College of Medicine and to the Graduate School and was 
accepted as partial fulfillment of the requirements for the 
degree of Doctor of Philosophy. 

August, 1996 


[Dean, College of Medicine 
Dean, Graduate School 

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